Nucleases are enzymes that cleave deoxyribonucleotides (DNA) or ribonucleotides (RNA) molecules, into smaller nucleotides fragments, e.g., oligonucleotides. Each enzyme recognizes a specific sequence of nucleotides, thereby cleaving the DNA or RNA at specific recognition sites. This sequence-specific cleavage is important in the production of smaller nucleotide fragments, gene isolation and sequencing, plasmid mapping, recombinant DNA manipulations, and the like. Unfortunately, however, the number of endonucleases is limited. Thus, chemically synthesized agents that cleave nucleotide sequences are important functional reagents for use in such molecular biology applications as well as in diagnostics and cancer therapy. They can be considered as artificial or synthetic nucleases.
Inorganic DNA cleaving reagents are one type of nucleotide cleaving agent of significant importance. The susceptibility of the ribose ring to oxidation has led to the use of a number of complexes that cleave DNA by oxidative mechanisms. Examples of such complexes include [Fe(EDTA)].sup.- (EDTA=ethylenediaminetetraacetic acid), [Cu(phen)].sup.+ (phen=1,10-phenanthroline), and metalloporphyrins, which are activated by H.sub.2 O.sub.2 or a reductant in the presence of O.sub.2. Another example is the photoactivated [Rh(phen).sub.3 ].sup.2+ complexes. See, for example, Tullius, Annu. Rev. Biophys. Chem., 18, 213-237 (1989); Dervan, Science 232, 464-471 (1986); Sigman et al., Accts. Chem. Res., 26, 98-104 (1993); Meunier, Chem. Rev., 92, 1411-1456 (1992); Pyle et al., J. Am. Chem. Soc., 111, 4520-4522 (1989); Uchida et al., Nucleic Acids Res., 17, 10259-10279 (1989). These cleaving agents are generally nonspecific. That is, they cleave DNA in a nonspecific manner, i.e., not at any one particular site or sequence.
The nonspecificity of the Fe(EDTA)-mediated cleavage has led to its use as a DNA footprinting reagent in identifying the binding locations of small molecules, such as proteins, antibiotics and other drugs, on DNA. That is, [Fe(EDTA)].sup.- is capable of being used to map the DNA binding location of small molecules because the bound molecule protects the DNA binding region from cleavage by the [Fe(EDTA)].sup.-.
Sequence specificity can be incorporated into these cleaving agents by tethering DNA recognition elements to the metal complex, a design principle analogous to that used by the antitumor drug bleomycin. See, for example, Dervan, cited supra.,; Sigman et al., cited supra.; Dervan et al., U.S. Pat. No. 4,665,184; and Stubbe et al., Chem. Rev., 87, 1107-1136 (1987). Bleomycin is a glycopeptide that binds to DNA and cleaves it at specific sites through the use of Fe.sup.+2 and O.sub.2 or Fe.sup.3+ and H.sub.2 O.sub.2. This principle involves the use of a DNA binding molecule to deliver a reactive metal complex to a specific sequence of nucleotides where cleavage occurs.
Whether site specific or not, the above-mentioned nucleotide cleaving agents operate oxidatively, thereby destroying a ribose ring to engender strand scission, i.e., cleavage. In this way the nucleotide sequence is not simply "cut" in such a way as to leave a 3' hydroxyl on one end and a 5' phosphate on the other, but the smaller pieces formed are effectively changed. This means the smaller pieces of DNA cannot be used in typical DNA cloning procedures, e.g., incorporation of a DNA fragment into a plasmid that can be amplified. Thus, the focus is shifting to the development of reagents that can cleave polynucleotides into fragments that can be readily cloned.
Hydrolytic reagents cleave polynucleotides through hydrolysis of the phosphate backbone as do restriction endonucleases in such a way as to leave a hydroxyl on one end and a phosphate on the other end. In this way the nucleotide sequence is simply "cut" into smaller pieces that can be used in typical cloning procedures. A number of metal ion complexes have been shown to hydrolyze phosphate esters as well as RNA with varying efficiencies; however, they are unable to hydrolyze the phosphate diester backbone of DNA. See, for example, Hendry et al., Prog. Inorg. Chem., 38, 201-258 (1990); DeRosch et al., Inorg. Chem., 29, 2409-2416 (1990); Modak et al., J. Am. Chem. Soc., 113, 283-291 (1991); Morrow et al., J. Am. Chem., 114, 1903-1905 (1992); Chin et al., J. Am. Chem. Soc., 111, 4103-4105 (1989); and Stern et al., J. Am. Chem. Soc., 112, 5357-5359(1990). The only reported metal-catalyzed DNA hydrolysis thus far is effected by Cu.sup.+2, Co.sup.+2, Zn.sup.+2, Cd.sup.+2, or Pb.sup.+2 complexed to a polyamine ligand tethered to a DNA binding [Ru(phen).sub.3 ].sup.2+ derivative, which afforded nicked (single stranded break) pBR322 at 37.degree. C. after 5-7 hours. Basile et al., J. Am. Chem. Soc., 109, 7550-7551(1987).
Thus, what is needed are effective agents that can cleave both DNA and RNA, single-stranded or double-stranded, particularly in the phosphate backbone in a manner in which the nucleotide sequence is cut to leave a hydroxyl on one end and a phosphate on the other end. Furthermore, what is needed are cleaving agents capable of cleaving nucleotide sequences in this manner at specific recognition sites.